Fig 1.
The pentose catabolic pathway (a), the Leloir pathway (b) and the oxido-reductive pathway (c) that convert arabinose, xylose and galactose.
The enzymes that catalyse particular metabolic steps in A. nidulans (white boxes), T. reesei (light grey boxes) and A. niger (dark grey boxes) are indicated. In the oxido-reductive pathway in A. nidulans the product of galactitol oxydation is most likely L-sorbose, while in A. niger and T. reesei this product was characterised as L-xylo-3-hexulose [13,14]. Unidentified or unconfirmed enzymes are marked with a star. Gene numbers of A. nidulans enzymes: LarA = AN7193, LadA = AN0942, LxrA = AN10169, XdhA = AN9064, XyrA = AN0423, XkiA = AN8790, GalE = AN4957.3, GalD = AN6182.3, GalF = 9148.3, GalG = AN4727.3, PgmB = AN2867.3, XxkA = AN7459. Gene numbers of A. niger enzymes: XyrA = An01g03740, LadB = An16g01710, XhrA = An16g01650, SdhA = An07g01290, HxkA = An02g14380.
Table 1.
A. nidulans strains used in this study.
Table 2.
Primers used in this study to generate the gene fragments for qRT-PCR analysis (a) and Southern Blot (b).
Fig 2.
Amino acid sequence alignment of A. nidulans GalR, AraR and XlnR.
The alignment was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo) [31] and visualized using Easy Sequencing in PostScript (http://espript.ibcp.fr/ESPript/ESPript/index.php) [32]. Conserved regions are marked by shaded boxes and similar regions by unshaded boxes. The Zn2Cys6 binuclear DNA binding domain is underlined and the six conserved cysteine residues are indicated by stars.
Fig 3.
Pairwise amino acid sequence alignment within the DNA binding domain of GalR, AraR and XlnR in A. nidulans.
The alignment was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo) [31] and visualized using Easy Sequencing in PostScript (http://espript.ibcp.fr/ESPript/ESPript/index.php) [32]. Conserved regions are marked by shaded boxes and similar regions by unshaded boxes.
Table 3.
Amino acid sequence identity (%) within the Zn2Cys6 binuclear DNA binding domain of GalR, AraR and XlnR in A. nidulans.
InuR is an example of non-paralogous TF.
Fig 4.
Growth profiling of an A. nidulans reference strain and single, double and triple disruptants of araR, xlnR, and galR on medium containing different carbon sources.
Strain codes are described in Table 1. Strains were grown for 3 days at 37°C.
Fig 5.
Expression levels of araR, xlnR and galR genes in ΔAraR, ΔXlnR and ΔGalR mutants in A. nidulans.
The expression was measured in the reference and single, double and triple regulatory mutant strains of A. nidulans after 2h of growth on D-fructose (black bars), L-arabinose (grey bars), D-xylose (striped bars) and D-galactose (checkered bars). The columns represent the mean and error bars represent standard deviation between biological replicates. Significant change in expression between the reference strain and the mutant was marked by the asterisk.
Fig 6.
Expression levels of six genes encoding enzymes involved in the PCP in A. nidulans.
The expression of larA, ladA, lxrA (a) and xyrA, xdhA, xki (b) was measured in the reference and regulatory mutant strains of A. nidulans after 2h of growth on D-fructose (black bars), L-arabinose (grey bars), D-xylose (striped bars) and D-galactose (checkered bars). The columns represent the mean and error bars represent standard deviation between biological replicates. Significant change in expression between the reference strain and the mutant was marked by the asterisk. The asterisk and the brace represent the significance between two indicated conditions.
Table 4.
Detailed regulation of genes involved in the PCP in A. nidulans.
Fig 7.
Expression of orthologous genes encoding enzymes involved in the Leloir (a) and the D-galactose oxido-reductive pathway (b) in A. niger.
The expression was measured in the reference and regulatory mutant strains of A. nidulans after 2h of growth on D-fructose (black bars) and D-galactose (checkered bars). The columns represent the mean and error bars represent standard deviation between biological replicates. Significant change in expression between the reference strain and the mutant was marked by the asterisk. Significant changes in expression between single and double mutants were marked by Greek symbols and braces: an alpha (α) represents a significant change between ΔA and ΔAΔG, a beta (β) represents a significant change between ΔG and ΔXΔG/ΔAΔG, a gamma (γ) represents a significant change between ΔX and ΔXΔG.
Fig 8.
Regulation of the oxido-reductive D-galactose catabolic pathway and the pentose catabolic pathway (PCP) in A. nidulans.
Genes of the PCP are regulated by the transcriptional activators XlnR (blue lines) and AraR (pink lines). Some of the D-galactose oxido-reductive genes are under regulatory control of GalR (green lines) together with XlnR and AraR. The expression of the hexokinase gene (hxkA) is observed in the A. nidulans ΔΔΔ triple mutant strain, probably due to the fact that this enzyme is also a part of glycolysis and therefore constitutively expressed. Some of the enzymatic conversions of the D-galactose oxido-reductive and the PCP pathway are verified, whereas others still need to be verified (marked by a star). Regulation of genes represented by solid lines is confirmed by expression analysis; dotted lines indicate that this regulation still needs to be verified. Dashed lines represent direct or indirect induction of TFs by monomer. Ortholog of ladB: AN4336, ortholog of red1: AN7914, ortholog of sdhA: AN2666.